Fig. 5

NtPsCRP2 and unconventionally secreted NtTCTP enter the ER and co-localize with the Golgi marker GmMan1 and exosome marker Ole1. a Confocal micrographs showing co-localization NtPsCRP2-GFP with the ER retention marker HDEL-mCherry with the exception of GFP foci (white arrowheads). Two-channel profile overlap (a-i). Emphasis on the lack of complete overlap (black arrowheads) of ER-HDEL with NtPsCRP2-GFP foci (a-ii). b NtPsCRP2 co-localization with the ER–Golgi vesicle marker GmMan1-mCherry from soybean. Frequency of co-localization (29 confocal slices, n = 340) (b-i). c Co-localization of unconventionally secreted NtTCTP with ER HDEL-mCherry. Visualization of ER co-localization of NtTCTP by two-channel pixel-intensity profiling (c-i). d NtTCTP also co-localizes with the ER–nuclear lamina and is present in the nucleoplasm. e NtTCTP co-localization with the Golgi vesicle marker GmMan1-mCherry. Arrowheads indicate granules formed by TCTP-GFP and those by GmMan1-mCherry. e–i Frequency of overlapping foci observed in 32 confocal slices (n = 292). f Dynamics of NtPsCRP2-GFP foci (green) and Golgi-derived vesicles (red) in the proximity of the plasma membrane. The top white arrows point to tethered NtPsCRP2-GFP foci over time, the red arrowheads Golgi vesicles alone, the green arrowheads NtPsCRP2-GFP foci alone, and the red-green arrowheads co-localized NtCRP1-Golgi signals, and foci 1–4 are additional NtCRP1-Golgi co-localized vesicles migrating clockwise towards the membrane horizon over time. Note the appearance and disappearance of co-localized NtPsCRP2-Golgi vesicles as well as NtPsCRP2-GFP foci alone. Scale bars = 20 μM. g NtTCTP co-localized with Ole1 in potential nanovesicle exosomes. Co-expression of NtTCTP-GFP with AtOle1-mRFP in tobacco leaf epidermal cells revealed granular co-localizations (marked with arrowheads). h Frequency of co-localizations observed from multiple leaf discs. Scale bars = 10 μM, RFP red fluorescent protein